General Considerations
Several aspects should be taken into consideration concerning the subject of the invention reported here, among which are the important advances in the fields of biochemistry, molecular biology, immunology and electrophysiology related to the knowledge generated about:    1)—the presence of integral proteins of biological membranes termed “ion-channels” playing a fundamental role in cellular communication, signal transduction pathways and general homeostasis of tissues and various organ functions;    2)—different levels of expression of these channels in cells of the immune system, mainly in T-lymphocytes, shown to play a clear role in events related to the onset of autoimmune diseases;    3)—the possible control of the channel function, hence possible treatment of disorders, through the addition of various chemicals, natural ligands and synthetically prepared substances, either reproducing the ligand as found in nature or preparing similar derivatives (peptidomimetics).
A multitude of potassium (K) channels have been discovered and reported to exist in the last fifteen years permitting their isolation, individual expression and functional analysis.
They are multimeric proteins implicated in the determination of cell membrane potential, thereby controlling smooth muscle tone, synaptic excitability, neurotransmitter release, and other processes. In this invention we want to emphasize the importance of a K channel species, sub-type Kv1.3, and its role in lymphocyte proliferation and in the control of autoimmune diseases by means of inhibiting this channel. This is a delayed-rectifier channel predominantly expressed in T lymphocytes [Grissmer et al., 1990; Lewis and Cahalan, 1995], different from the sub-types Kv1.1, Kv1.2 widely distributed in brain or Kv1.5 in heart tissue, to mention just a few of the sub-types of K channels.
The mechanisms by which modulation of Kv1.3 channels activity affects lymphocyte proliferation are being investigated in several laboratories and were the object of many recent publications (reviewed in [Beeton and Chandy, 2005; Judge and Bever, 2006, Panyi et al., 2006], including some patents (v.gr. U.S. Pat. No. 5,397,702 by Cahalan et al. 1995 and U.S. Pat. No. 6,077,680 by Kem et al. 2000).
Autoimmune diseases are known for their considerable worldwide morbidity. Among these diseases are: type-1 diabetes mellitus (insulin dependent), multiple sclerosis (MS), rheumatoid arthritis, Sjogren's syndrome, mixed connective tissue disease, systemic lupus erythematosus (SLE), myasthenia gravis, to mention just some of them. A relevant experimental model for autoimmune diseases is the experimental autoimmune encephalomyelitis (EAE). It is generally accepted that these autoimmune diseases result from the response of the immune system destroying specific tissues, either by a direct attack to the cells, or by producing auto-antibodies. The over-expression of Kv1.3 channels is a characteristic feature of autoreactive T cells thereby providing an excellent opportunity for the modification of their proliferation by blockers of Kv1.3.
In these lines of research and experimentation, several substances were described and even patented. One such example is the toxin ShK from the sea anemone Stichodactyla helianthus and several derivatives of it, claimed to have a protective effect against several autoimmune diseases (v.gr. U.S. Pat. No. 6,077,680 by Kem et al. 2000).
Among other natural ligands that are capable of affecting the function of ion-channels are toxic peptides isolated from scorpion venoms. K channel specific peptides isolated from these venoms are short-chain peptides containing 22 to 42 amino acids compacted by either three or four disulfide bridges. They are blockers of many different sub-types of channels, with a huge variability in selectivity and affinity (reviewed in [Giangiacomo et al., 2004; Rodriguez de la Vega and Possani, 2004]). For example, charybdotoxin is a potent blocker of Kv1.1, Kv1.2 and 1.3 Shaker type delayed rectifier channels but also blocks maxi-type K(Ca) channels [Rauer et al., 2000]. Margatoxin, another scorpion venom peptide, lacks K(Ca) channel blocking activity, but maintains a high affinity block of Kv1.3 channels. [Garcia-Calvo et al., 1993]. Agitoxin, noxiustoxin, kaliotoxin are examples of scorpion toxins that affect different types of K channels with distinct affinities and selectivities, but usually modify more than one sub-types of channels (recent reviewed in [Panyi et al., 2006]). Due to their relatively rigid three-dimensional structure, tightly maintained by disulfide bridges, some of these scorpion peptides have been used as “molecular calipers” for measuring distances between K channel amino acid residues in the outer vestibule of the channels [Krezel et al., 1995; Garcia et al., 2000]. The three dimensional structure of many scorpion toxins specific for K channels was resolved by nuclear magnetic resonance and/or X-ray diffraction methods, and in conjunction with the known structure of some K channels have provided the clue to model the interaction between the receptor (ion-channel) and the ligand (scorpion toxin). Site-directed mutagenesis of amino acid residues in both the ion-channels and the ligands has provided information for the identification of the putative interaction surface among this pair of receptor-ligand proteins [Rodriguez de la Vega et al., 2003]. This information is fundamental for the rational design of possible drugs with potential pharmacological applications. The only problem in using these naturally occurring peptides as potential drugs is the lack of specificity and affinity. At present, there are 20 sub-families of scorpion toxins, comprising over 125 structurally related peptides, classified by their sequence similarities and possible functions [Tytgat et al., 1999; Rodriguez de la Vega and Possani, 2004].
Molecular Basis for Kv1.3 Inhibitor-Based Therapy of Autoimmune Diseases
In this section the inventors present the state of the art knowledge on the control of several immunological diseases by simple application of ligands (peptides or organic compounds) capable of blocking with high affinity and high specificity the Kv1.3 ion-channels of “effector memory T-cells” (TEM) of lymphocytes.
It has been shown earlier that the mechanism by which Kv1 3 inhibitors interfere with the activation processes of lymphocytes evoked by physiological antigen stimulation or mitogens is the depolarization of the membrane and the consequent inhibition of the Ca2+ signal required for normal progression of the cell cycle to proliferation and production of the T-cell clones specific for a challenging antigen (reviewed in [Cahalan et al., 2001; Panyi et al., 2004; Panyi et al., 2006]). There are two types of K channels being responsible for maintaining a sufficiently hyperpolarized membrane potential (−50, −60 mV) [Verheugen et al., 1995] of T cells, the voltage-gated and depolarization activated channel denoted as Kv1.3 [Decoursey et al., 1984; Matteson and Deutsh, 1984]; and the Ca2+-activated K channel of intermediate conductance denoted as IKCa1 (or KCa3.1. according to a recent nomenclature) [Grissmer et al., 1993]. The activity of these channels provides the counterbalancing positive charge efflux required for the maintenance of a negative membrane potential during the influx of Ca2+ into the T cells through the Ca2+ release activated Ca2+ channels [Feske et al., 2006; Yeromin et al., 2006]. The contribution of these two types of K channels to the membrane potential of T cells depends on the activation status of the cells (resting vs. activated) and their functional role in the immune system determined by the degree of terminal differentiation of the T cells, as discussed below [Wulff et al., 2003].
Two types of T cells, the naïve and central memory T cells (TCM), require strong antigen stimulation and co-stimulation in peripheral (secondary) lymphoid organs to be activated. The naïve T cells that have not encountered previously an antigen bear CCR7+CD45RA+ functional marker expression. Central memory T cells (TCM, CCR7+CD45RA−), which cells mediate reactive memory, are probably arrested at intermediate stages of terminal differentiation to become effector memory cells (TEM) [Sallusto et al., 2004]. These cells have little or no effector function, but readily proliferate and differentiate to effector cells in response to antigenic stimulation. Protective memory is governed by effector memory TEM cells (CCR7−CD45RA+/−). TEM cells display characteristic sets of chemokine receptors and adhesion molecules that are required for homing to inflamed tissues where they exert immediate effector function. In several autoimmune diseases, including multiple sclerosis (MS) (Wulff et al., 2003), rheumatoid arthritis and type-I diabetes mellitus [Beeton et al., 2006], autoimmune psoriasis, lupus erythematosus, ulcerative colitis, sympathetic ophtalmia and bone resorption periodontal disease, chronically activated TEM cells are responsible for tissue damage, thus selective inhibition of the proliferation and functional activity of these cells is of utmost importance in the management of these diseases (reviewed in [Chandy et al., 2004; Beeton and Chandy, 2005; Panyi et al., 2006].
Resting human naïve, TCM and TEM of either CD4+ (helper) or CD8+ (cytotoxic) phenotype express similar number (200-300) of Kv1.3 and fewer than 30 IKCa1 channels per cell [Wulff et al., 2003]. Transformation of naïve and TCM cells to proliferating blast cells by specific antigen stimulation is accompanied by a modest (˜1.5-fold) increase in the number of Kv1.3 channels per cell, whereas the number of IKCa1 channels increase dramatically (500 channel/cell) and thus, they acquire an IKCa1highKv1.3low ion channel phenotype. In contrast, activation of TEM of either CD4+ or CD8+ phenotype in the peripheral tissues is accompanied by a dramatic increase in the number of Kv1.3 channels to ˜1500/cell without any change in the IKCa1 level thereby the channel phenotype of activated TEM becomes IKCa1lowKv1.3high.
The causal link between Kv1.3high TEM and autoimmune disorders is substantiated by the following data obtained in human diseases:    1) myelin-reactive T cells from the peripheral blood of MS patients are Kv1.3high [Wulff et al., 2003];    2) myelin-reactive T cells from the peripheral blood of healthy controls are Kv1.3low, consistent with a naïve/TCM phenotype;    3) stimulation of MS patient T cells with irrelevant antigens such as insulin peptide, ovalbumin or with conventional mitogens did not induce the generation of TEM with Kv1.3highIKCa1low channel phenotype;    4) Kv1.3high TEM cells were shown in postmortem MS brain inflammatory infiltrates and in the parenchyma of demyelinated MS lesions [Rus et al., 2005];    5) T cells isolated from the synovial fluid of human patients suffering from Rheumatoid Arthritis (RA) express large amounts of Kv1.3 as compared to T cells of the same donor but isolated from peripheral blood. These Kv1.3high T cells were CCR7− indicating that they are TEM cells [Beeton et al., 2006];    6) Short term antigen specific CD4+ T cell lines (TCLs) generated from peripheral blood lymphocytes of Type 1 Diabetes Mellitus (T1DM) human patients and specific for T1DM-associated autoantigens insulin and GAD65 display the characteristic features of TEM cells including the lack of CCR7 antigen (CCR7−) and Kv1.3high channel phenotype [Beeton et al., 2006].
As the membrane potential control of TEM cells is exclusively governed by the activity of Kv1.3 channels, the proliferation of these cells, their functional activity, and thus the symptoms of the autoimmune disease, should be ameliorated by the use of Kv1.3 inhibitors. The following in vitro and in vivo data in the literature support this scenario:    1) In vitro proliferation of chronically activated human T cell lines bearing the characteristics of TEM (CCR7−, Kv1.3high) and specific for myelin antigen [Wulff et al., 2003] or TEM cells isolated from the synovial fluid of RA patients is completely and permanently suppressed by Kv1.3 specific blockers peptide such as ShK [Wulff et al., 2003], ShK(L5) or by the small-molecule Kv1.3 blocker PAP-1 [Beeton et al., 2006];    2) In vivo experiments with Margatoxin, another high affinity Kv1.3 blocker peptide, showed that block of Kv1.3 leads to the inhibition of delayed-type hypersensitivity reactions in miniswine; this reaction is a good measure of the activity of effector memory T cells [Koo et al., 1997];    3) Treatment of MBP-specific rat T cells with ShK or ShK-Dap22 during their in vitro stimulation with MBP (sensitization phase) along with repeated application of the peptides into the recipient animals (during the effector phase) prevented the adoptive transfer of Experimental Autoimmune Encephalomyleitis (AT-EAE) into Lewis rats [Beeton et al., 2001]. EAE in rats [Ben Nun and Cohen, 1982], is the best characterized model for the human disease MS characterized by similar pathogenesis and neurological abnormalities, and the disease causing T cell population is the myelin-specific TEM having Kv1.3high channel phenotype. The combined application of Kv1.3 and IKCa1 channel blockers also ameliorated the symptoms of EAE when administered following its onset [Beeton et al., 2001];    4) Pristane-induced MHC class II-restricted chronic arthritis model (PIA) in Dark Agouti rats is a rat model for the human disease Rheumatoid Arthritis. Single daily injections of ShK(L5) significantly reduced the number of joints affected by the disease during the trial period (up to 34 days) [Beeton et al., 2006];    5) The efficacy of a Kv1.3 inhibitor to prevent experimental autoimmune diabetes (EAD, a rat model for T1DM of humans) was studied in MHC class II-restricted DP-BB/W rats [Beeton et al., 2006]. It was shown that repeated daily administration of PAP-1, a high affinity and selectivity small-molecule blocker of Kv1.3 reduced the fraction of rats showing the symptoms of EAD by ˜50% (assayed at 110 days of age) as compared to control animals treated with vehicle only. This was accompanied by a decreased intraislet T cell and macrophage infiltration and reduced β cell destruction in the PAP-1-treated group as compared with vehicle-treated control (assayed between 35-70 days of age) [Beeton et al., 2006].
The inhibition of T cell proliferation by Kv1.3-specific inhibitors is specific to TEM cells, which makes these compounds ideal tools for the management or prevention of autoimmune diseases. Although antigen-induced proliferation of resting naïve and TCM cells is partially sensitive to Kv1.3-mediated inhibition, the transcriptional up-regulation of IKCa1 channels overcomes this in pre-activated T cells and renders the proliferation of these cells to be sensitive to IKCa1 inhibitors but not to Kv1.3 inhibitors [Ghanshani et al., 2000]. This restricted action of Kv1.3 and IKCa1 inhibitors on different T cell subsets underlies the importance of the selectivity of a given molecule for Kv1.3 over IKCa1. It was also shown recently that the inhibition of TEM proliferation by Kv1.3 inhibitors can be overcome by excessive antigen stimulation mimicking the activation of TEM cells by pathogens and vaccine antigens during normal protective memory immune reactions [Beeton et al., 2006]. Thus, the application of high affinity and high selectivity Kv1.3 inhibitors ideally targets TEM cells repeatedly activated during autoimmune reactions whereas leave other protective functions of the immune system unaltered.
In addition to human T and B lymphocytes Kv1.3 channels are also expressed in several organs and tissues (including the central nervous system, kidney, liver, skeletal muscle), and the block of Kv1.3 channels in the cells may give rise considerable side effects. Extensive in vitro and acute and chronic in vivo toxicological tests were performed previously for ShK(L5) [Beeton et al., 2005; Beeton et al., 2006] from the peptide blockers group and for PAP-1 [Schmitz et al., 2005; Beeton et al., 2006] from the group of small molecule blockers. These studies showed the lack of clinical symptoms for neurological and cardiac side effects or histopathological changes in tissues where Kv1.3 is expressed. Thus, the beneficial treatment-effects of Kv1.3 blockers listed above combined with minimal or the complete absence of side effects point towards the applicability of selective Kv1.3 blockers in the management of autoimmune diseases.
In summary, data above suggest a critical role of Kv1.3 K channels in the execution of a physiological immune response, and point to the applicability of a therapeutic intervention in autoimmune disease by the inhibition of Kv1.3 channels.